DESCRIPTION: (Applicant's Abstract) Electroneutral Na-Cl, Na-K-2Cl, and K-Cl cotransporters participate in the movement of salt and fluid across epithelia and play a major role in the maintenance and regulation of the volume of a wide variety of cells. While these transporters have been studied extensively during the past 15 years, it is only recently that their molecular identity has been revealed. We have recently shown that two of these cotransporters, the Na-K-2Cl cotransporter and the K-Cl cotransporter, are expressed highly in the brain. Their role in the nervous system is incompletely understood. Growing evidence suggests that besides playing an important role in regulating the composition of the CSF, they may also play a major role in neuronal function. In fact, Na-K-2CI cotransporter expression in neurons has been suggested to determine the effect of GABA on membrane potential, which in turn may be important during neuronal maturation and response to neuronal injury. This proposal is aimed at understanding the physiology of these cotransporters in neuronal cells. Through detailed quantitative studies we will investigate 1) the role of the secretory Na-K-2Cl cotransporter (BSC2), an inward transport mechanism, and the neuronal-specific K-Cl cotransporter (KCC2), an outward transport mechanism, in establishing the steady-state intracellular Cl- concentration in neurons, and 2) the developmental regulation of these two cotransporter mechanisms. These studies, by involving functional and molecular approaches, will lead to a better understanding of the role of these electroneutral cation-chloride cotransporters in neuronal function. Regulation of intracellular and extracellular ion composition has important implications for the physiology and pathophysiology of the brain. For instance, regulation of intracellular Cl- concentration is crucial for controlling the function of GABA in the CNS. The GABA response in turn is an important feature of the normal function of the neuron, e.g. for the control of slow wave sleep in the thalamus, or the control of diurnal rhythmicity in the hypothalamus, a critical feature of neuronal plasticity during development, and a critical feature in various pathologies such as cerebral trauma, cerebral ischemia, where neuronal damage involves changes in GABA-induced excitability properties, potentially due to significant changes in intracellular Cl- concentration.